This invention is in the field of chromatographic support media. The invention itself is directed to support media which is nonporous to the extent that it is impenetrable by solutes, particularly macromolecular solutes, and to methods of preparing such media from porous starting materials.
High-performance liquid chromatography (HPLC) is widely used for analytical and preparative separations of biopolymers. Columns for HPLC generally use smaller and more rigid beads as stationary phase supports than do columns for conventional, low-pressure chromatography. Silica, for example, is a commonly used stationary phase in HPLC. Agarose beads, on the other hand, are more common in low-pressure chromatography. Agarose beads can be adapted for use in HPLC by being made smaller (3-10.mu. diameter) and more rigid (Hjerten, S. et al., J. Chromatogr. 215, 137 (1981); Hjerten, S., Acta Chem. Scand. B36, 203 (1982); Hjerten, S., et al., J. Chromatogr. 296, 115 (1984), but this is done by a procedure which is expensive and laborious. Furthermore, the resulting beads are still compressible to some extent, and compression considerably increases the flow resistance around the beads, which is undesirable in some cases. A maximum linear velocity of 3 cm/min for a 30 cm long molecular-sieve chromatography column, for example, has been reported by Hjerten, S., et al., J. Chromatogr. 316, 301 (1984).
In some cases, compressibility of the stationary phase is desirable since it decreases the distance between individual beads. Relatively large beads, which are easy to prepare, can thus be given chromatographic properties equivalent to smaller beads simply by being compressed.
When compressibility is combined with a porosity which is sufficiently low that the beads are effectively nonporous--i.e., any remaining pores are so small or so narrow as to be substantially impermeable to the solutes in the sample being separated--further advantages are achieved. With the solutes no longer diffusing into and out of the beads, the chromatographic interactions can be restricted to the external surfaces of the beads, resulting in sharper resolutions and faster flow rates.